A Thesis


to the Faculty of

California State University, Chico


In Partial Fulfillment

of the Requirements for the Degree

Master of Science





Lindsey Kaylee Wallace

Fall 2012



Lindsey Kaylee Wallace

Fall 2012


______Eun K. Park, Ph.D.


______Gordon Wolfe, Ph.D., Committee Chair

______David Keller, Ph.D.

______Daniel Edwards, Ph.D.


This work was supported by NSF grant EFRI-0938157. I would like to thank

Mark Brown, from Iowa State University, for GC-MS analysis of PULCA components.

Katie Scott for help with radio labeling experiments in the Summer of 2012. Thanks to

Sarah Hoddick who provided positive feedback, immense emotional support, and death metal. I also owe appreciation to my committee members, Dr. Edwards and Dr. Keller, for their guidance and assistance that made my thesis possible. Most of all, thank you to Dr.

Wolfe for not only the opportunity to work in his lab but also continued guidance and support throughout my project.




Acknowledgments……………………………………………………………………. iii

List of Tables………………………………………………………………………..... vi

List of Figures……………………………………………………………………….... vii

Abstract……………………………………………………………………………...... ix


I. Introduction………………………………………………………………. 1

E. huxleyi: A Globally-Important ……………………………… 1 E. huxleyi Neutral Lipids……………………………………………... 1 PULCA Accumulation and Cell Stress………………………………. 5 Biofuel Applications…………………………………………………. 6 PULCA Localization…………………………………………………. 7 Possible Biosynthetic Pathways…………………………………….... 8 Study Objectives, Questions, and Hypothesis………………………... 12

II. Materials and Methods………………………………………………….... 15

Cultures……………………………………………………………….. 15 Nile Red Staining, Microscopy, and Fluorometry……………………. 16 Lipid Extractions……………………………………………………... 17 Thin Layer Chromatography…………………………………………. 18 Gas Chromatography – Mass Spectrometry Analysis………………... 18


CHAPTER PAGE Radioisotope Uptake Experiments…………………………………..... 20

III. Results…..……………………………………………………………….... 23

GC-MS Analysis of Neutral Lipids…………………………………… 23 Tracing Carbon Flow Into Neutral Lipids Over a Growth Cycle…...... 29 Carbon Flow Into Neutral Lipids Over Light-Dark Manipulations…... 34 Effect of Inhibitors on Carbon Flow Into Neutral Lipids………...... 40

IV. Discussion……………………………………………………………….... 45

Identification of Neutral Lipids with GC-MS………………………... 45 Changes in Production of Neutral Lipid Types Based on Growth Conditions……………………...... 46 Acetate Utilization……………………………………………………. 47 Bicarbonate Utilization……………………………………………...... 48 Inhibitor Effects on Neutral Lipid Synthesis…………………………. 49 Summary of Findings…………………………………………………. 51

Possible PULCA and C31-33 cis-alkene Synthesis Pathways………….. 52

References…………………………………………………………………………...... 55




1. Variations in LC alkenes in E. huxleyi strains showing presence of

C31-33 cis-alkenes and/or C37-39 trans-alkenes…………………….. 4

2. Provasolli-Guillard Culture Collection (NCMP) algal cultures……….... 16

3. Inhibitors used in pulse radio labeling experiments…………………...... 20

4. Major hydrocarbon types identified in I. galbana (1323) and E. huxleyi

strains using GC-MS analysis…………………………………….. 26

5. Comparison of bicarbonate vs. acetate uptake………………………….. 41

6. Summary of early inhibitor effects on 14C-acetate uptake and

14C-bicarbonate into CCMP 1516………………………………… 42

7. Summary of inhibitor effects on 14C-acetate uptake into CCMP 1516…. 43

8. Summary of inhibitor effects on 14C bicarbonate uptake into

CCMP 1516 and 3268 ………………………………………………. 44




1. PULCA skeletons with diunsaturated trans bonds at w14,21...... 2

2. C31-33 LC alkene skeletons with cis geometry………………………… 3

3. Biosynthetic pathways proposed by Rontani et al. (2006)…………... 10

4. A model of polyunsaturated FA (PUFA) and PULCA biosynthesis ... 12

5. Diagram of lipid extraction procedure ………………………………. 22

6. Separation of nonpolar lipids using TLC and then identification of

lipids with GC-MS……………………………………………….. 25

7. Growth of 1516 and 3268 over 14 days……………………………… 28

8. Pools of neutral lipids in exponential vs. stationary with bicarbonate

E. huxleyi cells…………………………………………………..... 29

9. Cell growth of cultures for pulse chase experiment………………….. 31

10. Pulse chase radio labeling of E. huxleyi strain 1516 with 14C Acetate. 32

11. Pulse chase radio labeling of E. huxleyi strain 1516 with

14C bicarbonate…………………………………………………… 33

12. Light-dark experiment schematic……………………………………... 35

13. Neutral lipid per cell over light-dark cycle………………………...... 35

14. Neutral lipid pools per cell over light-dark cycle………………...... 37

15. Light effects 14C bicarbonate uptake……………………………...... 39



16. 14C acetate pulse labeling in the dark, and testing for bacterial

controls...... 40

17. TLC plate of hydrocarbon, neutral lipid, and PULCA extraction

method...... 42







Lindsey Kaylee Wallace

Master of Science in Biology

California State University, Chico

Fall 2012

Emiliania huxleyi and some related prymnesiophyte algae produce a novel group of polyunsaturated long-chain C37-39 alkenones, alkenoates, and alkenes as their major neutral lipids, however their biosynthesis pathway is unknown. Like triglycerides, these lipids are believed to be utilized as storage lipids and are accumulated in lipid bodies to be used as a fuel source, presumably. Also C31-33 cis-alkenes have been identified in E. huxleyi but are believed to be different in synthesis and function. By studying the synthesis of these lipids I set out to discern how these two types of neutral lipids are formed in E. huxleyi. I used a combination of techniques, including GC-MS analysis, radiolabeling, and inhibitors, to examine lipid pools during growth cycles, bicarbonate dosing, and light-dark manipulations. By using GC-MS analysis I identified


the presence of C31-33 cis-alkenes exclusively in E. huxleyi strains CCMP 1516 and 371, and galbania strain CCMP 1323; where E. huxleyi strains CCMP 1742, 3266, and 3268 had both cis-alkenes and C37-38 trans-alkenes. I also found accumulation of both cis-alkenes and C37-38 trans-alkenes in light to dark manipulations was similar to that seen in storage lipids, suggesting that the long chain cis-alkenes are in fact another storage lipid and may share a similar synthesis pathway to the trans-alkenes. Using radiolabeling studies I found external acetate is not acquired under light dependent mechanisms and is utilized primarily in production of polar lipids; while external bicarbonate acquisition and use in lipid synthesis is light-dependent, and as a cellular building block it is distributed more evenly amongst lipid pools. Also, flow of carbon into CCMP 1516 and 3268 cells from external bicarbonate into lipid pools is inhibited by cerulenin (fatty acid synthase inhibitor), flufenacet (elongase inhibitor), and quizaloflop

(acetyl-CoA carboxylase inhibitor). Platensimycin (fatty acid synthase II inhibitor) only affects flow of bicarbonate into CCMP 1516 C31-33 cis-alkenes. Finally, flow of carbon into CCMP 1516 cells from external acetate into lipid pools is inhibited by cerulenin.

Flufenacet and quizaloflop only affects flow of acetate into C37-39 trans-alkenes.

Platensimycin has no effect on the flow of acetate into lipid pools in CCMP 1516.

Though these results show precursors of acetate and bicarbonate are used in the synthesis of these neutral lipids, and through inhibitor studies I have identified many mechanisms vital to their synthesis, the biosynthetic pathway is yet unclear.




Emiliania huxleyi: A Globally-Important Algae

Emiliania huxleyi is a marine haptophyte algae, found ubiquitously in oceans.

This single celled is typically 3-7 µm in diameter and has a triphasic cycle. The complex life cycle of E. huxleyi involves several stages with distinct cell types: the -bearing diploid motile cells, vegetative diploid naked cells, and haploid motile scale-bearing swarm cells; each stage can exist independently and reproduce (Laguna et al 2001, Rokitta et al 2011). They form a large part of marine biomass and are the most abundant of the coccolithophorids. Coccolithophorids are organisms that produce , which are calcium carbonate disks that are extruded to the surface of the cell, their function is still unknown. E. huxleyi can accumulate in large white blooms observable from space as dense as 107 cells per L (Olson & Strom 2002), the white color is due to the shedding of coccoliths produced by the cells. E. huxleyi’s ability to produce coccoliths and abundance makes them an important part of the biological carbon cycle and contributor to atmospheric composition (Herfort et al 2002,

Tsuji et al 2009).

E. huxleyi Neutral Lipids

Most algae produce triglycerides as neutral lipids, but E. huxleyi and other members of the order produce unique polyunsaturated long chain (LC)



C37-39 lipids including alkenes, alkenones, and alkenoates [abbreviated as PULCA

(Eltgroth et al 2005)]. Unlike cis-polyunsaturated fatty acids (FA) that we normally find in membranes, PULCA have 2-4 unusual trans-alkene bonds that occur at 7-C intervals (fig.1). However, the trans configuration has only been confirmed for C37 alkenone, and C37-38 alkenes (de Leeuw et al 1980, Rechka & Maxwell 1988a, Rechka &

Maxwell 1988b, Rieley et al 1998). Bonds of di-unsaturated alkenones, C37-40, had a fixed double bond position at ∆14,21 from the carbonyl group, contrasting with earlier speculation. Di-unsaturated shorter chain C35-36 ketones found in E. huxleyi CCMP 1742, exhibit double bonds at ∆15,22 and ∆16,23 suggesting an alternate pathway of synthesis

(Rontani et al 2006). Using a reduction-silylation technique, trace amounts of monounsaturated alkenones, C35-38, were found in samples from several , including E. huxleyi, with double bond positions different than polyunsaturated analogues

(Rontani et al 2004, Rontani et al 2001).

Figure 1: PULCA skeletons with diunsaturated trans bonds at ∆14, 21. (a) C36 methyl or ethyl alkenoates, (b) C37-alkene, (c) C37 methyl alkenones, and (d) C38 ethyl alkenones.

These haptophytes also have another set of C31–33 alkenes that have been determined to have cis geometry, and may be biosynthetically distinct from PULCA (fig.

2). E. huxleyi strains vary in the type alkenes produced (table 1) (Conte et al 1995,

Rieley et al 1998). C31-33 alkenes also vary in double bond arrangement with ∆15,22 bonds (Rontani et al 2004).

∆1, 22 ∆2, 22 Figure 2: C31-33 LC alkene skeletons with cis geometry. (a) C31:2( ), (b) C31:2( ), (c) C31:2 (∆3, 22), (d) C33:2(∆1, 22), and (e) C33:2(∆2, 22).


Table 1: Variations in LC alkenes in E. huxleyi strains showing presence of C31-33 cis- alkenes and/or C37-39 trans-alkenes, modified from Conte et al. 1995.

E. huxleyi Strains Origin C31-33 Alkenes C37-39 Alkenes 5/90/25g NE Atlantic, Temperate + - 88COCCO Gulf of Maine + - 88E Gulf of Maine + - English Channel/North + - 92D Sea English Channel/North + + 92E Sea B92/11 Norwegian Fjords + - B92/131 Norwegian Fjords + - B92/21 Norwegian Fjords + - B92/317 Norwegian Fjords + - English Channel/North + + CH25/90 Sea DW53/74/6 NE Atlantic, Subtropical + - DW60/3/8 NE Atlantic, Iceland Basin + - DW61/12/6 NE Atlantic, Iceland Basin + + DW61/3/16 NE Atlantic, Iceland Basin + + DW61/67/5 NE Atlantic, Iceland Basin + + DW61/87/17 NE Atlantic, Iceland Basin + + EH2 NW Pacific, Subtropical + - G1779Ge NE Atlantic, Iceland Basin + - M181 Sargasso Sea + - S.AFRICA Indian Ocean, Subtropical + - VAN55 NE Pacific, Temperate + + VAN556 NE Pacific, Temperate + +

The ratio of di- to tri-unsaturated C37 methyl alkenones, denoted Uk'37, has been shown to vary linearly with seawater temperature and has proven to be a useful indicator of past sea surface temperature in sediments (Conte et al 1995). At colder growth temperatures, PULCA are more polyunsaturated (Brassell et al 1986, Conte et al

1995, Conte et al 1998, Prahl & Wakeham 1987). However, the calibration of unsaturation index depends on strain genetics (Conte et al 1998) and environmental factors such as nutrient limitation or light availability (Epstein et al 2001, Epstein et al


1998, Prahl et al 2003, Versteegh et al 2001, Yamamoto et al 2000). Analysis of

Chrysotila lamellisa HAP 17, a related haptophyte, which produces only C31-33 cis- alkenes, shows a trend of more unsaturated C31 alkenes with decreasing temperature, the same trend seen in PULCA (Rontani et al 2004). PULCA's unique structure indicates a different physiological and biochemical role than other FA normally produced in organisms (Eltgroth et al 2005).

PULCA Accumulation and Cell Stress

These long chain (LC) lipids are the primary storage lipids of the algae, which produce very little triacylglycerol (TAG) (Bell & Pond 1996, Marlowe et al 1984a,

Marlowe et al 1984b). While E. huxleyi is in stationary growth, PULCA shows a pattern of high accumulation, which closely resembles storage of triglycerides (Sawada &

Shiraiwa 2004). A 10% - 20% increase of PULCA accumulation under nitrogen or phosphorous limitation was observed from total cell carbon levels in stationary phase

(Epstein et al 2001, Prahl et al 2003). Nutrient limitation coupled with excessive amounts of bicarbonate augmentation also result in massive amounts of PULCA synthesis (Wolfe & Erlendson in preparation). It was also observed, that under prolonged darkness, PULCA stores decrease due to catabolism (Prahl et al 2003).

PULCA accumulation has been found to be dependent on environmental conditions as well as culture age. Under nitrogen deprivation, E. huxleyi maintains significant photosystem II (PSII) function for at least 38 days (Loebl et al 2010). Many photosynthetic organisms cannot continue functioning once they become nitrogen limited, nitrogen is a key component for the maintenance of the abundant proteins


associated with the photosynthetic apparatus because (PSII) reaction centers are constantly damaged by light energy (photoinactivation), cells need nitrogen to synthesize the new proteins required for PSII repair. Conversely, when E. huxleyi becomes phosphorous limited PSII function declines within 7 days (Loebl et al 2010). Iron is also a key component in PULCA synthesis, like light limitations iron is necessary for during inorganic carbon fixation in photosystems I and II and various cytochromes of the photosynthetic electron transport chain. Iron limitation thus affects all processes, and results in diminished cell growth and lipid accumulation (Schulz et al

2007). Radiolabel studies using 14C bicarbonate have shown that as concentrations of dissolved inorganic carbon (DIC) increase, so does bicarbonate uptake for both photosynthesis and calcification (Herfort et al 2002).

Biofuels Applications

The unusual structure of PULCA and formation under nutrient deplete conditions, suggests that these lipids might be used as potential biofuel precursors. The majority of lipid based biofuel studies have focused on TAG production, the typical storage lipid of most photosynthetic algae. However, TAGs are not ideal energy sources because the glycerol must be removed via alkaline transesterification. Also, most algal

TAGs are polyunsaturated and highly susceptible to photooxidation during storage

(Greenwell et al 2010, Schenk et al 2008). PULCA, on the other hand, are essentially pure hydrocarbon, and lack the glycerol unit found in TAGs (Wolfe & Erlendson in preparation). Pyrolysis, a method being developed for producing fuel from biomass, of

PULCA shows high energy yield values (Qingyu et al 1999). PULCA are also more


resistant to photooxidation during storage than TAGs (Rontani et al 1997). Algal biofuel production has been limited by the inability to produce massive cell growth coupled with high lipid content, but since nutrient limitations and bicarbonate feeding both stimulate

PULCA production E. huxleyi is a more cost effective organism for biofuels (Rosenberg et al 2008).

PULCA Localization

Originally, it was suggested that PULCA were used for structural purposes involved in maintaining membrane fluidity, meaning they were localized primarily to the cell membrane (Prahl et al 1988). A later study by Sawada and Shiraiwa (2004), using cell fractionation, supported this finding when PULCA were found associated with cell membranes and organelles. PULCA were predominantly reported to be in the and coccolith producing compartment with low amounts found in fractions (Sawada & Shiraiwa 2004). However Conte and Eglinton (1993) could find no association of PULCA with membrane components, and it was hypothesized that trans bonds would be less stabilizing than cis bonds and thus would not be used in structural support (Mouzdahir et al 2001). typically store TAGs in lipid bodies (Murphy 2001), and many algae package their neutral lipids into cytoplasmic vesicles or lipid bodies. Eltgroth et al.(2005) has shown that lipid bodies in E. huxleyi increased under nutrient limitation, and then disappeared during prolonged darkness, which was supported by a previous study done on Isochrysis galbanea (Liu & Lin 2001).

These lipid vesicles have been purified by subcellular fractionation and revealed to contain mostly PULCA (Wolfe & Erlendson in preparation). Early studies using electron


microscopy have suggested lipid body vesicles exist freely in the cytoplasm and are not attached directly to any other organelle function (Mouzdahir et al 2001); more recently

PULCA isolated in lipid bodies have been found associated or in , as well as the cytoplasm (Eltgroth et al 2005). Further analysis of lipid droplets is now being conducted using Nile Red staining, a fluorescent lipophilic dye, which allows better imaging of lipid droplets and allows simple quantification (Wolfe & Erlendson in preparation).

Possible Biosynthetic Pathways

In most , FA synthesis occurs in the via a heteromeric fatty acid synthesis (FAS-II, ‘prokaryote’ form), composed of discreet ketoacyl synthases, dehydratases, keto and enoyl reductases, and thioesterases. This pathway generates palmitate and oleate, which are then esterified to acetyl-CoA and exported to the endoplasmic reticulum (ER) for further elongation. Elongation and desaturation of acyl-

CoA esters, occurs by a complex series of ER-bound elongases. These elongated acyl-

CoA esters are precursors to polyunsaturated FAs (PUFA) of membrane phospholipids and glycolipids, as well as sphingolipids. Orthologs to ER-bound elongases have been identified in the Emiliania genome and align well with other elongases known to produce

PUFA and sphingolipids (Wolfe & Erlendson in preparation). In non-photosynthetic eukaryotes, FAs are produced by cytosolic fatty acid type I synthases (FAS-I), large homomeric enzyme complexes that add malonyl or methylmalonyl coupled to acyl carrier proteins (ACP) to acyl-CoA esters. Based on PULCA structures and the discovery of novel compounds in Black Sea sediments, as well as an apparently mutated


E. huxleyi strain, Rontani et al. (2006) proposed a de novo biosynthesis pathway by which the C37-39 skeletons are built from acetate or propionate starters (fig. 3). They also proposed that like epicuticular waxes, which mainly consist of straight-chain aliphatic hydrocarbons (Koo et al 2005, Leide et al 2007), PULCA skeletons then undergo either an acyl reduction pathway, which yields alkenoates, or a decarbonylation pathway, to yield ketones, which are then reduced to alkenes. C31-33 cis-alkenes, which are believed to be originated from oleic acid, and its believed function as a structural lipid

(Bowsher et al 2008, Rontani et al 2006, Templier et al 1987); a hypothesized lipid synthesis pathway shows formation from oleic acid using typical wax ester synthesis found in plants (fig. 3b).


Figure 3: Proposed model for synthesis of long chain neutral lipids in E. huxleyi, modified from Rontani et al (2006). (a) Pathway for generation of alkenone and alkenoate carbon skeleton from acetyl-SCoA primer, and (b) pathway for synthesis of cis and trans alkenes.


While Rontani et al. (2006) did not discriminate among plastidial FAS-II and cytosolic FAS-I systems, Wolfe & Erlendson (in preparation) found proteins for homomeric ACCase, malonyl-CoA:ACP transacylase, and several FAS-I homologs associated with E. huxleyi lipid bodies, suggesting a cytosolic location, and further support of an FAS-I system is found in the observed inhibition of PULCA biosynthesis under cerulenin, a specific inhibitor of type I β-ketoacyl-ACP synthase (Sorrosa 2005).

Additionally, some long-chain FAs are synthesized via polyketide synthase (PKS) systems, which has been found in both prokaryotes and eukaryotes (Napier 2002); including other marine haptophytes and chromalveolates () (Manning &

Claire 2010, Monroe & Dolah 2008). PKS resemble FAS synthesizing activities, but utilize a production-line array, and are able to produce a wide variety of products ranging from keto, hydroxyl, enoyl, or alkyl functionality groups, with even- and odd-chain lengths and branched structures. In the Emiliania genome at least ten loci have been identified homologous to PKS models, and three were identified during lipid proteome tests by Wolfe and Erlendson Sub (2010). However, it is not clear if PULCAs are synthesized de novo from a FAS-I or PKS-I system, or if C14-18 FAs synthesized in by FAS-II are exported to the cytosol and modified by FAS-I or PKS-I systems, as suggested by Wolfe & Erlendson (in preparation); (fig. 5). .


Figure 4: A model of polyunsaturated FA (PUFA) and PULCA biosynthesis, which both compete for acyl-CoA pools exported from the . PUFA and sphingolipids (blue arrows) are created by ER bound elongase systems that use malonyl-CoA, while PULCA and related hydrocarbons (red arrows) are hypothesized to originate from FAS/PKS systems that utilize malonyl-ACP. Enzymes shown in red have been found in lipid body and lipid body-ER proteome extractions by Wolfe and Erlendson Sub (2010). Abbreviations: ACC = acetyl CoA carboxylase; ACS = acetyl CoA synthetase; AS = acyl synthetase; FAS-I = fatty acid synthase I; MCAT = malonyl- CoA:ACP transacylase; PD = pyruvate dehydrogenase; PKS = polyketide synthase; TE = thioesterase (Wolfe & Erlendson in preparation).

Study Objectives, Questions, and Hypothesis

In this study, the metabolic flux of PUFA and PULCA accumulation in E. huxleyi was monitored using radiolabeled precursors, 14C bicarbonate or acetate. GC-MS


was used in identifying lipids from E. huxleyi. Cultures were analyzed under different life stages and cycles, in the presence of inhibitors, dependence on light, and strain types.

My Questions

1. How do E. huxleyi strains differ in production of neutral lipids? How do these

components change over growth phases, bicarbonate dosing, and light-dark


2. How does inorganic (bicarbonate) and organic (acetate) carbon get incorporated

into PULCA and how does this change in exponential and stationary growth


3. How do inhibitors affect the incorporation of inorganic and organic carbon into

lipid pools?

4. Do these results support or refute the pathways proposed by Rontani et al (2006),

or evidence for plastidial vs. cytosolic pathways?

My Hypotheses

1. Variations in LC cis-alkene and LC trans-alkene distribution is dependent on


2. Acetate is utilized primarily in polar lipids and is independent of light conditions.

3. Bicarbonate is utilized equally by cell components and is dependent on light.

4. PULCA Alkenes use the same mechanism as alkenoates and alkenones in their

synthesis while cis- C31-33 alkenes are synthesized by an alternative method.


5. PULCA synthesis occurs via de novo fatty acid synthesis in the cytoplasm,

utilizing a FAS-I or PKS-I system.





E. huxleyi and I. galbana cultures (table 2) were obtained from Provasoli-

Guillard National Center for Culture of Marine Phytoplankton (NCMP), formally the

CCMP, and maintained nonaxenically in the Wolfe Lab since 2009. I grew cultures in

100-1000 mL volumes of artificial sea salts (Sigma-Aldrich, St. Louis, MO) with added f/2 nutrients (AlgaBoost, AusAqua, Australia) under 100-200 µE m-2s-1 cool-white illumination at 16 ± 1⁰C in a 16 hour light: 8 hour dark diurnal cycle. To simulate cells incubated in darkness, I wrapped flasks in foil; all other conditions remained the same. I determined culture density and growth phases by using a haemocytometer.



Table 2: Provasolli-Guillard Culture Collection (NCMP) algal cultures used in this study (Rokitta et al 2011).

CCMP Growth Synonyms Origin Latitude Notes Strain# (⁰C) Emiliania huxleyi North 371 12-1 Atlantic, 32N 62W 22 - 26 High calcifying Sargasso Sea Sequenced 2008 CCMP2090, by Department of 1516 AC665, South Pacific 2.7S 83W 18 - 22 Energy through RCC1242 the Joint Genome Institute Non-calcifying, CCMP2758, calibration 1742 North Pacific 50N 145W 11 - 16 NEPCC55A standard for paleothermometry AC472, High calcifying, 3266 RCC1216, Tasman Sea 42S 170E 11 - 16 diploid version of TQ26 3268 Non-calcifying, RCC1217, 3268 Tasman Sea 42S 170E 11 - 16 haploid version of TQ26(N) 3266

Isochrysis galbana

ISO, Strain 1323 Irish Sea 54 N 4.8 W 11 - 16 Non-calcifying “I”, NEPCC2

Nile Red Staining, Microscopy, and Fluorometry

I stained cells with 1 µg/ mL Nile Red in DMSO for 15 min, after which I centrifuged cells at 10,621 x g for 1 – 2 min to concentrate for viewing. Wet mounts were viewed with an Olympus BX51 microscope (Melville, NY, USA) and a Pixera

Penguin 600ES CCD camera (Los Gatos, CA, USA) under fluorescence using blue


excitation (455 nm). A Schott glass bandpass output filter at 495 nm (Oriel optics

#51720, Stratford, CT, USA) was added to block chlorophyll emission. I performed

Fluorometry using a BioTek Synergy HT platereader on 200 µL aliquots of cells to determine cell density using 485/20 nm excitation and 540/20 nm emission filters, chlorophyll using 485/20 nm excitation and 697/30 nm emission filters; and lipids after

Nile Red staining using dual fluorescence: 485/20 nm excitation, 590/20 nm and 645/30 nm emission filters.

I tested cultures for phosphate by taking 1 mL samples, centrifuging and removing cells, the remaining supernatant was then assayed with a phosphate low range molybdate assay kit (L147240, Orbeco, FL) and read on a platereader at 880 nm.

Lipid Extractions

I collected cells onto 2.4 cm GF/F glass microfiber filter (Whatman, Florham

Park, NJ, USA) and washed them by running approximately 5 mL I/O through the filter.

Then, I placed filters into vials containing 9.8 mL of 10:5:4 MeOH:CHCl3:H2O giving an initial one phase system. Vials were vortexed vigorously and incubated for at least 24 hrs in the dark. 2.6 mL of CHCl3 and 2.6 mL of H2O to the vial to form a 10:5:4

CHCl3:MeOH:H2O final two phase Bligh Dyer system (Bligh & Dyer 1959). I vortexed the vials and spun them in a clinical centrifuge at 2,581 x g for 10 min. The top phase is the aqueous phase while the lower phase is the organic phase. I removed the organic phase into a glass collection tube, and dried under N2 and heat. The extraction was repeated by adding back CHCl3 to the vial two more times. I re-suspended dried lipids in

20-50 µL of 2:1 CHCl3:MeOH and stored at -20 ºC.


Thin Layer Chromatography (TLC)

Silica gel TLC plates (Whatman, Florham Park, NJ, USA) were activated with solvent 1 (MeOAc : IPA : CHCl3 : MeOH : 0.25% KCl, 25:25:25:10:9) and then baked at

120⁰C for 20 min. I pipetted samples or applied them with glass capillaries 0.8 cm from the bottom of the plate. Polar lipids were separated using solvent 1. Neutral lipids were separated using solvent 2 (hexane : diethyl ether : glacial acetic acid, 80:20:2). I visualized lipids by dipping the plate in 10% cupric sulfate in 8% H3PO4 for 2 min and then charring at 160⁰C. Lipids that I recovered for further tests were developed by first staining the plates with Nile Red (12.5 ug/ mL in MeOH : H20, 80:20) and then visualized under UV light.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

Cultures were sampled for GC-MS analysis; I tested variations in strains, growth stages, and under excess sodium bicarbonate (10 mM). I collected 1.00E+9 cells on GF/A 47 mm glass microfiber filter (Whatman, Florham Park, NJ, USA), 200 mL per filter. I placed filters in 6 mL of 1:1 hexane:CHCl3, and vortexed vigorously. The sample was then incubated at room temperature overnight, after which I then centrifuged the sample at 5,125 x g for 10 min. I then removed the solvent, containing lipids, to a tube containing 2.4 g of silica. I pre-treated the silica by washing in hexane three times, to remove any impurities. I pipetted 6 mL of 1:1 hexane:CHCl3 onto the original filter, vortexed, and centrifuged at 5,125 x g for 10 min. Again I removed the solvent to the new tube containing silica. Finally, I added 1 mL of 1:1 hexane:CHCl3 to the filter, and centrifuged without vortexing at 5,125 x g for 5 min, the solvent was added to the silica


tube. I then centrifuged the silica tube at 5,125 x g for 10 min, the solvent, containing neutral lipids, was removed and blown down with N2. I added 4 mL of 1:1 hexane:CHCl3 to the silica, then I vortexed, and centrifuged at 2,581 x g for 5 min; I removed the solvent once again and blew down the sample with N2, this was repeated one more time. Samples were kept plastic free, and glass was muffled at 500 ºC for at least 4 hrs.

I re-suspended samples in 1 mL 1:1 hexane:CHCl3 and applied them to silica gel TLC plates. Neutral lipids were separated using solvent 2 (hexane : diethyl ether : glacial acetic acid, 80:20:2). I approximated lipids by charring a separate standard, run with the TLC plate. The standard was visualized by staining with 3% cupric acetate 8% phosphoric acid for 2 min, and charred at 160 ºC for approximately 5 min. Based on the standard I then scrapped separately three bands labeled hydrocarbons, ethyl ketones, and methyl ketones. I extracted the lipids from the scrapings using 2 mL of 1:1 hexane:CHCl3, vortexed, and centrifuged at 5,204 x g for 5 min. I removed the supernatant and placed it in a 1.5 mL GC/MS vial, then removed the solvent with N2.

The samples were kept at -20 ºC, and then sent to Iowa State for GC-MS analysis.

GC-MS was performed using a (HP 6890 GC with HP 5973 MSD) instrument equipped with a HP-5MS 5% Phenyl Methylpolysiloxane capillary GC column (30 m,

250 µm film, 0.25 µm i.d; Agilent, United States) using helium gas as a carrier. The temperature program was 60 °C for 0 min, then 30 °C/min to 130 °C for 0 min, then 4

°C/min to 300 °C for 20 min. MS settings were undertaken in electron impact (EI) mode


(70eV, scan time 1 s; m/z 50 to 300). Quantification of lipids was achieved using 50 µg of n-heatriacontane as an internal standard per sample.

Radioisotope Uptake Experiments

I performed pulse labeling or pulse chase labeling on E. huxleyi cells with

0.25 µCi/ ml of [1-14C] sodium acetate, 0.25 µCi/ ml of [1,2-14C] sodium acetate, or with

0.5 µCi/ ml of [14C] sodium bicarbonate under varying conditions. Cells were labeled at different cell densities, different growth phases, different strain types, in the presence or absence of light, in excess sodium bicarbonate (10 mM), with bacterial controls, and in the presence of inhibitors. Bacterial controls I tested were 10 µL/ mL kanimycin and ampicillin applied for 30 min or centrifugation at 1000 x g for 10 min. Inhibitors that I applied were 0.5-2 hrs before labeling; Table 3 identifies inhibitor concentrations and function. I took samples at varying time points from the pulse and lipids extracted and fractionated.

Table 3: Inhibitors used in pulse radio labeling experiments. All inhibitors were dissolved in dimethyl sulfoxide (DMSO).

Inhibitor Target Final (µM) Citation Cerulenin FAS-I 25 (Sorrosa 2005) Flufenacet Elongase 50 (Tucci et al 2010) Platensimycin FAS-II 10 (Wang et al 2006) Quizalofop ACCase 200 (Zuther et al 1999)

For pulse chase labeling experiments, I filtered samples onto 2.4 cm GF/F glass microfiber filter (Whatman, Florham Park, NJ, USA) and washed them by running approximately 5 mL I/O through the filter. Filters were placed into vials containing 9.8


mL of 10:5:4 MeOH:CHCl3:H2O giving an initial one phase system. I vortexed vials vigorously and incubated for at least 24 hrs in the dark. 2.6 mL of CHCl3 and 2.6 mL of

H2O to the vial to form a 10:5:4 CHCl3:MeOH:H2O final two phase Bligh Dyer system

(Bligh & Dyer 1959). The vials were vortexed and spun in a clinical centrifuge at 1,652 x g for 10 min. The top phase was the aqueous phase while the lower phase is the organic phase. The organic phase was removed into a glass collection tube, and dried under N2 and heat. I repeated the extraction by adding back CHCl3 to the vial two more times. Dried lipids were then re-suspended in 20-50 µL of 2:1 CHCl3:MeOH and stored at -20 ºC. I obtained further separation by applying samples to TLC plates, using the method described above. TLC plate coating containing samples was removed with a razor blade, and the samples were counted in Snyder scintillation cocktail (Snyder &

Stephens 1962).

For the pulse experiments, I collected 2 mL of cells via pelleting and re- suspended in 1 mL of Bligh Dyer 10:5:4 MeOH:CHCl3:H2O solution. At least 24 hrs later I added 250 µL of CHCl3 and 250 µL of H2O, vortexed, and then centrifuged at

20,817 x g for 2 min. The organic phase was removed and blown down with N2 gas, this was repeated two more times by adding back 500 µL of CHCl3. Samples were re- suspend in 1 mL of hexane. I then transferred the 1 mL sample to a tube containing 200 mg of silica, then vortexed and spun at 20,817 x g for 2 min. Solvent was then removed, which contained hydrocarbons; this step was repeated by re-applying 500 µL of hexane at least 2 times. Then 500 µL of 1:1 hexane:CHCl3 was applied to the silica, vortexed and centrifuged at 20,817 x g for 2 min. Solvent was then removed extracting the PULCA, again the extraction was repeated at least 2 times. In some experiments I extracted


PULCA and hydrocarbons together by skipping the 1 mL hexane step. Finally 500 µL of methane was applied to the silica, vortexed and centrifuged at 20,817 x g for 2 min. The solvent was removed extracting polar lipids; the extraction was repeated 2 more times.

Lipids removed were put into 10 - 20 mL of ScintiVerse (Fisher Scientific) and radioactivity was determined using a Beckman LS 6000LL scintillation counter using external quench correction (fig. 5).

Figure 5: Diagram of lipid extraction procedure.



GC-MS Analysis of Neutral Lipids

Development of Extraction for GC-MS, and Analysis of Strains

I developed a method for separating the neutral lipids by silica gel fractionation into hydrocarbons and alkenones & alkenoates (fig. 6a). Extracted lipids were analyzed by GC-MS and structures confirmed by mass and mass fragment analysis.

While all cultures showed C37-39 alkenones and alkenoates, I observed that some strains also produced hydrocarbons of these chain lengths, while other strains produced almost exclusively C31-33 hydrocarbons (fig. 6, table 4). C31-33 hydrocarbons contained MS spectra resembling findings by Rontani et al. (2004, 2006), and thus is assumed to be cis- alkenes since trans-alkenes of C31-33 length have never been found. This confirms prior observations that there are two classes of long-chain neutral hydrocarbons in E. huxleyi.

In contrast, Rieley et al. (1998) showed that the C37-39 hydrocarbons were PULCA-types

(polyunsaturated with trans bonds, containing similar positional desaturations as the alkenones and alkenoates). Separation and GC-MS analysis of what we call the ethyl ketone (EK) band on TLC plates revealed that this region also contains methyl esters.

Additionally I saw that the abundance of PUCLA appeared strain dependent, CCMP

1323, 371, 1742, and 3268 made large quantities of alkenes compared to CCMP 1516 and 3266; CCMP 3266 produced very low levels of all neutral lipids (table 4). Though



GC-MS results showed low levels of methyl ketones, methyl esters, and ethyl ketones; however TLC plates show an abundance of these lipids comparable to alkenes (table 4, fig. 6a).


S MK EK HC MK EK HC b E. huxleyi 3268 Hydrocarbons

C36 Standard




E. huxleyi 3268 Hydrocarbons b C36 Standard C38:3 C31:2 C37:2

E. huxleyi 371 Hydrocarbons c C31:2 C36 Standard

Figure 6: Separation of nonpolar lipids using TLC and then identification of lipids with GC-MS. (a) Samples were first separated on TLC, scraped individual bands from the TLC and then re-extraced lipids from the TLC. Plate displayed shows separation and purity of the process. Each band was analyzed with GC-MS. Standard (S) contains 10 µg of C36 and 25 µg of cholesterol,bands for hydrocarbons (HC), ethyl ketones and methyl esters (EK), and methyl ketones (MK). (b) Shows hydrocarbon sample from strain 3268; which was found to contain large amounts of C38:3 alkenes, C37:2 alkenes, and trace amouts of C31:2 alkenes. C36 standard added was 50 µg. (c) Hydrocarbon sample from strain 371; which contains C31:3 and C31:2 alkenes, and 50 µg

C36 standard.


Table 4: Major neutral lipid types identified in I. galbana (1323) and E. huxleyi strains using GC-MS analysis. x denotes abundance less than 50 µg standard. X denotes abundance approximately equal to 50 µg standard, XX denotes abundance greater than 50 µg standard. Strain  1323 1516 371 1742 3266 3268


C31-33 XX x X x x x

C37-38 XX x XX


C37-38 MK x x x x x x

C37-38 EK x x x x x

C37-38 ME x x x x x

Changes In Neutral Lipids Over A Growth Cycle

Based on the analysis of strain neutral lipid variation, I compared two strains over a growth cycle and following bicarbonate treatment (fig. 7a), to compare the response of C31-34 and

C37-38 hydrocarbons. CCMP 1516 represented the C31-33 hydrocarbon group, and CCMP 3268 the C37-38 group. In both strains, I observed an increase in lipid production once cells entered stationary phase with the addition of bicarbonate (fig. 7b), similar to the observations of Wolfe and Erlendson (sub-2010). Cells stained with NR were observed under the microscope with an emission filter that removed red fluorescence, blocking polar lipids. Nile Red staining showed large yellow-staining of membranes and lipid bodies in cultures once they reached day 14, however at day 5 no neutral lipids were visible. I found at day 5 there were no visible neutral lipids in the cells, compared to day 14 where yellow-staining of membranes and lipid bodies was observed indicating the presence of large amounts of neutral lipids in the cells (fig. 7c-f).


GC-MS analysis of CCMP 1516 showed a slight increase of 2.68 x 10-8 in PULCA lipids and C31-33 hydrocarbons once cells reached stationary phase with bicarbonate stimulus, but the ratio of lipid types to one another remained the same (fig. 8a). I observed in CCMP 3268 that

GC-MS analysis of different growth phases showed an increase of 2.26 x 10-7 between exponential to stationary with bicarbonate stimulus cells (fig. 8b), in general the production of lipids compared to CCMP 1516 was far greater in CCMP 3268.




E. huxleyi 1516

1.E+06 E. huxleyi 3268 Cells/mL

1.E+05 0 7 14

b 1516 3268 8000

4000 F485/590 nm F485/590 0 0 7 14 Days

c d d

Figure 7: Growth of 1516 and 3268 over 14 days. (a) Log of cell growth. At day 11, 10 mM NaHCO3 was added (arrow). Bars indicate the sampling of exponential cells (day 5), and stationary cells (day 14). (b) Neutral lipid concentrations of cultures, determined with NR staining. (c, d) Composite images of day 14 bicarbonate-dosed cells stained with NR (c: 1516; d: 3268). Images were collected at 1/20s with emission bandpass filter to eliminate red fluorescence at 1/20s.


a 3.00E-07 b 3.00E-07

Hydrocarbons: C31:3, Alkenes C31:2, C33:3, and C33:2 Methyl Ketone Methyl Ketone Ethyl Ketone and Methyl 2.00E-07 2.00E-07 Ethyl Ketone and


Methyl Esters

g lipid/cellg

g lipid/cellg

µ µ 1.00E-07 1.00E-07

0.00E+00 0.00E+00 Expo Stat-BC Expo Stat-BC

Figure 8: Pools of neutral lipids in exponential (Expo) vs. stationary with bicarbonate (Stat- BC) E. huxleyi cells determined with GC-MS analysis. (a) strain 1516, and (b) strain 3268.

Tracing Carbon Flow Into Neutral Lipids Over a Growth Cycle

Using pulse chase radio labeling with 14C bicarbonate or acetate I was able to monitor the flow of carbon into neutral lipids over a growth cycle. CCMP 1516 was grown over a period of 14 days, cell counts were taken periodically to track growth of the culture (fig 9a). On day 5 and day 14 portions of the culture were used for radio labeling experiments, at the same time media was tested for phosphate concentrations. At day 5 during exponential phase cultures had a high level of phosphate, while at day 14, the cultures were phosphate starved (fig. 9a). After labeling lipids I extracted and then separated them using TLC, plates were then scrapped as labeled in figure 9b to isolate the different neutral lipid pools. I scraped glycolipids (GL) from the TLC plate in figure 9b as nonpolar lipids because they migrated with Solvent 2.

Pulse chase 14C acetate labeling over a period of 23 hrs with cells in darkness from

6.5-18.5 hrs showed the label was taken up more by stationary phase cells, and primarily

30 incorporated into polar lipids, there appeared to be no significant impact of the dark cycle on acetate incorporation (fig. 10). In neutral lipids of exponential cells, I found the 14C acetate label went highest into ethyl ketones (EK), followed by GL, methyl ketones (MK), and hydrocarbons

(HC) last. However in stationary cell neutral lipids, label went highest into glycolipids, and then

EK, MK, and HC (fig. 10e, f).

Pulse chase 14C bicarbonate labeling over a period of 23 hrs with cells in darkness from 6.5-18.5 hrs showed the label was taken up more by exponential phase cells, and incorporated into polar lipids and neutral lipids more equally than the ratio seen in 14C acetate labeling (fig. 11). Decrease in lipids in stationary phase appears due to a change in polar lipid incorporation (fig. 11c, d). Also CCMP 1516 cells took up 14C acetate more readily than 14C bicarbonate overall. In neutral lipids of exponential and stationary cells, I found the 14C bicarbonate label went highest into GL, MK, EK, and HC last (fig. 11e, f).




Figure 9: Cell growth of cultures for pulse chase experiment. (a) Growth of cells, phosphate limitation. (b) TLC plate used to sepearte out neutral lipids for pulse chase experiment, lipids visualized by Nile Red staining of plate and UV fluorescence. Standard contains PE, PG, C, TAG, FAME. Each sample 1-8 contains hydrocarbons (HC), ethyl ketones (EK), methyl ketones (MK), glycolipids (U), and polar lipids (P) highlighted in boxes.


Exponential Phase Cells Stationary Phase Cells 300000 Total 300000

a Aqueous b


] Organic

Ci Ci µ µ 200000 200000


100000 100000 C [DPM C /

C [DPM C / Aqueous 14

14 Organic Fixed Fixed Fixed Fixed 0 0 0 6 12 18 24 0 6 12 18 24 Time (Hrs) Time (Hrs) Total Lipid Total Lipid

c Polar d Polar


200000 Nonpolar ] 200000 Nonpolar



µ µ

100000 100000




14 Fixed Fixed 0 Fixed 0 0 6 12 18 24 0 6 12 18 24 Time (Hrs) Time (Hrs) EK EK MK GL f HC GL


6000 ] 6000

] MK


Ci µ

µ HC

4000 4000

C C [DPM /

C [DPMC / 14

14 2000 2000

Fixed Fixed Fixed 0 0 0 6 12 18 24 0 6 12 18 24 Time (Hrs) Time (Hrs) Figure 10: Pulse chase radio labeling of E. huxleyi strain 1516 with 14C acetate. Samples of 2.4x10-9 cells were taken at each time point and then lipids extracted and quantified. Grey box represents dark period. Error bars represent standard deviation for two samples. (a), (b) Incorporation of acetate into cells (total), and partitioning by Bligh-Dyer extration into organic and aqueous phases. (c), (d) Incorporation of acetate into lipid components, a combination of nonpolar lipids and polar lipids. (e), (f) Incorporation of acetate into nonpolar lipids: hydrocarbons (HC), ethyl ketones (EK), methyl ketones (MK), and glycolipids (GL).


Exponential Phase Cells Stationary Phase Cells 20000 Total 20000 Total

a Organic b Organic

] Aqueous ] Aqueous

Ci 15000 15000


µ µ

10000 10000 C [DPMC / 5000 C [DPM /

14 5000 14

Fixed Fixed 0 0 Fixed 0 6 12 18 24 0 6 12 18 24 Time (Hrs) Time (Hrs)

10000 Total Lipid 10000 Total Lipid

c Polar d Polar


8000 Nonpolar ] 8000 Nonpolar



µ µ 6000 6000

4000 4000


C [DPMC / 14

2000 14 2000 Fixed Fixed 0 Fixed 0 0 6 12 18 24 0 6 12 18 24 Time (Hrs) Time (Hrs)

6000 GL 6000 GL

e HC f HC

5000 EK 5000 ] ] EK


Ci MK µ µ 4000 4000 3000 3000

2000 2000


C [DPM / C 14

14 1000 1000 Fixed Fixed Fixed Fixed 0 0 0 6 12 18 24 0 6 12 18 24 Time (Hrs) Time (Hrs) Figure 11: Pulse chase radio labeling of E. huxleyi strain 1516 with 14C bicarbonate. Samples of 2.4x10-9 cells were taken at each time point and then lipids extracted and quantified. Grey box represents dark period. Error bars represent standard deviation for two samples. (a), (b) Incorporation of bicarbonate into cells (total), and partitioning by Bligh-Dyer extration into organic and aqueous phases. (c, d) Incorporation of bicarbonate into lipid components, nonpolar lipids and polar lipids. (e, f) Incorporation of bicarbonate into nonpolar lipids: hydrocarbons (HC), ethyl ketones (EK), methyl ketones (MK), and glycolipids (GL).


Carbon Flow Into Neutral Lipids Over Light-Dark Manipulations

Neutral Lipid Accumulation In Light Versus Dark

To study the effects of darkness on neutral lipid biosynthesis, and the interactions between bicarbonate feeding and darkness, I employed the following manipulation (fig. 12).

Cells were grown to stationary phase, CCMP 1516 had a density of 6.3x10-6 cells/mL and CCMP

3268 had a density of 5.5x106 cells/mL, then taken from their light:dark cycle and placed continuously in the dark for 3 days, after which a lipid sample was taken for GC-MS analysis.

Cells were then placed back in the normal light:dark cycle for 6 days, on the 6th day another lipid sample was taken for GC-MS analysis. The culture on the 6th day was then split into what I called flask #2 and #3 and excess bicarbonate was added to both. Flask #2 was placed in the dark and flask #3 was left in the light for another 3 days, after which I then took another lipid sample for analysis. On the 9th day I swapped the flasks placing flask #2 in the light, and flask

#3 in the dark. On day 12 I took one more sample for GC-MS analysis.


Figure 12: Light-dark experiment schematic. Suns represent cultures in their regular light:dark cycle, moons represent cultures in permanent darkness. Test tubes show when samples were taken. Day 6 samples were split into two new flasks and 10 mM of bicarbonate were added to each. I found using Nile Red analysis of cultures, cells in the dark began to lose neutral lipids, and once they were returned to the light regained them. With the addition of bicarbonate neutral lipids greatly increased in cells that were in the light, however cells in the dark did not create more neutral lipids in the presence of bicarbonate (fig. 13).


a b

4.00E-03 4.00E-03

2.00E-03 2.00E-03

0.00E+00 0.00E+00

0 3 6 9 12 Nile Red Fluorescence (F485/590) Nile Red Fluorescence (F485/590) 0 3 6 9 12 Days Days Figure 13: Neutral lipid per cell over light-dark cycle, as assayed by Nile Red staining (a: 1516, b: 3268). Yellow symbols indicate treatments in light; black in dark. Arrow shows addition of 10 mM sodium bicarbonate on day 6, when flasks were split. Analysis of Lipid Pools During Light-Dark Manipulations

The neutral lipids I extracted were analyzed by GC-MS and from that analysis I found that like the Nile Red results, neutral lipid pools also fluctuated. Both CCMP 1516 and 3268 cultures were dominated by alkenes (fig. 14), with PULCA in far less abundance (not shown).

Alkenes, C31-33, in CCMP 1516 decreased when cells went into the dark, and then increased when cells were in the light, also the addition of bicarbonate at day 6 caused the amount of nonpolar lipids being produced to increase dramatically (fig. 14a, b). Alkenes, C31-33 and C37-39, in CCMP 3268 were also dependent on light conditions and showed the same fluctuations as seen in CCMP 1516 (fig. 14c, d). A comparison of total C31-33 alkenes to C37-39 alkenes in CCMP

3268 showed that while the amount of alkenes increased the ratio of alkene types fluctuated. Day

0 to 6 cultures were dominated by C37-39 hydrocarbons (fig. 14e, f); but between day 6 to 12 cells going from dark to light favored C31-33 hydrocarbons (fig. 14e), and cells going from light to dark showed a decrease in the ratio of C37-39 hydrocarbons that then began increasing once cells went back into the dark (fig. 14f).


a b

6.0E-07 6.0E-07

4.0E-07 4.0E-07 C31-34 HCs C31-34 HCs

2.0E-07 2.0E-07

µg lipid/ lipid/ µg cell

µg lipid/ lipid/ µg cell CCMP 1516 CCMP

0.0E+00 0.0E+00 0 3 6 9 12 0 3 6 9 12 Days Days 1.5E-07 1.5E-07 c C31-34 HCs d

C37-38 HCs

1.0E-07 1.0E-07 C31-34 HCs C37-38 HCs

5.0E-08 5.0E-08

µg lipid/ lipid/ µg cell

µg lipid/ lipid/ µg cell

0.0E+00 0.0E+00 0 3 6 9 12 0 3 6 9 12 Days Days

e 100% f 100%


75% 75% %HC as C31-34 %HC as C31-34 50% 50% %HC as C37-38 %HC as C37-38

25% 25%

%Total Alkenes %Total Alkenes 0% 0% 0 3 6 9 12 0 3 6 9 12 Days Days

Figure 14: Neutral lipid pools per cell over light-dark cycle, as assayed by GC-MS. (a, b) Alkenes, C31-33 in CCMP 1516 (c, d) Alkenes, C31-33 and C36-38 in CCMP 3268. (e, f) % of total alkenes in CCMP 3268. Carbon Flow Into Neutral Lipids During Light-Dark Manipulations

Using pulse labeling, I studied uptake of 14C bicarbonate and 14C acetate into cells in light vs. darkness treatments in five experiments (not all shown). I developed a rapid method of fractionating activity (see Materials & Methods). I Bligh-Dyer extracted fractionated cells into organic and aqueous fractions, which were each counted and compared to the total cell uptake.


Organic fractions were then fractionated in vitro on silica gel into polar and non-polar fractions.

In general, recoveries of polar and nonpolar fractions added to total organic, suggesting losses were minimal.

I found that light is critical to the uptake of 14C bicarbonate. Cells pre-treated in the dark were unable to incorporate any bicarbonate as long as darkness persisted. Figure 15 shows examples for 1 or 2 hr darkness, but this was also true of longer time periods (not shown). Once cells were returned to the light, they immediately started to take up the label. I also found that when cells in the light were placed into darkness, bicarbonate incorporation stopped (fig. 15c).

During the 1 hr dark period, it appeared that some label was lost from the aqueous fraction (fig.

15c). This label did not move into other fractions, and it is possible that fixed triose phosphates were mobilized for respiration to support ATP synthesis during darkness. When cells were placed back into the light, uptake immediately resumed.


a 30% Light b 8%

Total Cell

Dark → Light Nonpolar 6%

20% Polar Added/ Added/ mL]

4% DPM


C [% C

C [% DPM C Added/ mL] 14

14 2%

Fixed Fixed Fixed Fixed

0% 0% 0 1 2 3 4 0.5 1 1.5 2 Time (Hrs) Time (Hrs) 3.0%

c TC

Aqueous Organic 2.0% Nonpolar Polar


C [% DPM C Added/ mL]

14 Fixed Fixed 0.0% 0 0.5 1 1.5 2 2.5 Time (Hrs) Figure 15: Effects of light on 14C bicarbonate uptake. Grey boxes represent darkness. (a) Uptake into total cells, either in the light or in the dark  light, shaded region represents time of dark. (b) Uptake into different cell components, shaded region represents time of dark. (c) Uptake into cell components from light, to dark, and then back again; shaded region represents time of dark. Error bars represent standard deviations for two samples. In contrast, 14C acetate labeling showed only weak dependence on light. CCMP 1516 cells pre-incubated in the dark immediately took up 14C acetate (fig. 16a), although rates may have slowed during the dark period. To examine whether incorporation of acetate in the dark might be due to bacterial contaminants, I tested several methods of reducing bacterial activity, including differential centrifugation or addition of antibiotics. In both cases, acetate was still


incorporated into at a level similar to the untreated control (fig. 16b), and uptake in dark and in light were nearly similar. As a further check that my methods to reduce bacterial activity did not

affect E. huxleyi cells, I also tested 14C bicarbonate uptake, and found this was both light-

dependent and unaffected by antibiotics, suggesting minimal injury to algae (not shown).

a 60% Light 6% Control

Dark → Light b Antibiotic


40% 4%

Added/ Added/ mL] DPM

20% DPMAdded/ mL] 2%

C [%C

C [% C

14 14

0% 0% Fixed Fixed Fixed 0 1 2 3 4 0 1 2 Time (Hrs) Time (Hrs)

Figure 16: 14C acetate pulse labeling in the dark, and testing for bacterial controls. Shaded region represents period of darkness. (a) Labeling cells from light  dark, versus light control showing total cell uptake. (b) Label uptake into cells in the dark  light with bacterial controls implemented. Effect of Inhibitors on Carbon Flow Into Neutral Lipids

I observed that bicarbonate and acetate consistently showed different partitioning into different pools. Over 1-4 hr incubations, about 50% of bicarbonate partitioned into organic

(lipid) pools, compared to only 35% of acetate (table 5). However, the acetate incorporated into the organic fraction overwhelming partitioned into polar, rather than neutral lipids (table 5).

Bicarbonate labeling of polar and neutral lipids was variable, but consistently produced higher fractions of nonpolar lipids than acetate. This suggests the flow of carbon from both labels occurs via different pathways.


Table 5: Comparison of bicarbonate vs. acetate uptake. Observation / Label bicarbonate acetate

Light-dependent uptake? yes No*

Label into organic in dark? no yes

% uptake as organic ~50% 35%

% organic as NP 20-40% 10%

*may be masked by bacterial contaminants.

To further explore these differences, I used specific inhibitors of different lipid processes to try and understand carbon flow into neutral lipid. Early studies involved pulse labeling with

14C-acetate or 14C- bicarbonate in the presence of inhibitors (table 6). Inhibitors were only applied 30 min prior to pulse labeling. I found that in the presence of cerulenin bicarbonate uptake into nonpolar lipids was affected, and acetate incorporation in polar lipids was slightly inhibited. Quizaloflop appeared to inhibited incorporation of bicarbonate into both nonpolar and polar lipids, and flufenacet appeared to increase flow of bicarbonate into nonpolar lipids. Results obtained had good recovery and linearity, and represent two experiments.


Table 6: Summary of early inhibitor effects on 14C-acetate (AC) uptake and 14C- bicarbonate (BC) into CCMP 1516 E. huxleyi cell lipids. Cells were pretreated with inhibitors for 30 min prior to pulse labeling. Nonpolar Polar Lipids

Inhibitor Target AC BC AC BC

Cerulenin FAS-I, II - ++ + -

Flufenacet Elongase - I - -

Platensimycin FAS-II - - - -

Quizaloflop ACCase - +++ - +++

“+” denotes degree of inhibition: +++ > 75%, ++ 50-75%, and + 25-50% “-“ had no affect, > 0-25%

I refined my fractionation protocol to separate hydrocarbons and alkenones/alkenoates, to compare uptake into strains making both C31-33 and C37-39 HCs (table 4).

Tests with unlabeled cells showed I was able to separate the majority of hydrocarbons, alkenones/alkenoates, and polar lipids did not contaminate the neutral lipids (fig. 17).

1 2 3 4 5 6 7 8 S

Figure 17: TLC plate of hydrocarbon, neutral lipid, and PULCA extraction method. Lane 1 and 5 contained sample of total lipids unseparated, Lane 2 and 6 contained hydrocarbons separated, Lane 3 and 7 contained remaining neutral lipids, Lane 4 and 8 contained polar lipids. Standard (S) contained C36, oleic acid, and cholesterol (10 µg).


I found that 14C acetate incorporation was affected by cerulenin in all lipid pools and quizaloflop only impaired label accumulation in PULCA, but had no effect on 1516 C31-33 alkenes (table 7).

Experiments were conducted several times on CCMP 1516 and results showed linearity over a 2 hour period with R2 values greater than 0.95. It appeared that flufenacet inhibited PULCA slightly and caused a slight increase in alkene and polar label acquisition, suggesting a re-routing of carbon flow.

Table 7: Summary of inhibitor effects on 14C-acetate uptake into CCMP 1516 E. huxleyi cells. Cells were pretreated with inhibitors for 2 hrs prior to pulse labeling. Inhibitor Target Alkenes PULCA Polar Lipids

Cerulenin FAS-I, II ++ +++ +++

Flufenacet Elongase I + -

Platensimycin FAS-II I - I

Quizaloflop ACCase - +++ +

“+” denotes degree of inhibition: +++ > 75%, ++ 50-75%, and + 25-50% “-“ had no affect, and is comparable to control. “I” increase in label greater than 10% of control When pulse lableing with 14C bicarbonate CCMP 1516 and CCMP 3268 with inhibitor treatments, I found both strains were affected similarly in carbon flow to neutral lipids but there were slight variations in polar lipids (table 8). Experiments were conducted several times on CCMP 1516 but only once on CCMP 3268, and uptake was linear (R2>0.95) over 2 hr.

Cerulenin, flufenacet, and quizalofop decreased carbon flow into alkenes and PULCA equally in both strains. In CCMP 1516 polar lipids I found that cerulenin caused only a slight decrease compaired to other inhibitors, and in in CCMP 3268 cerulenin appeared to increase carbon flow into polar lipids. Also in pulse labeling experiments on CCMP 1516 it was found that platensimycin affected carbon flow into alkenes but not PULCA or polar lipids, CCMP 3268 was not tested (table 8).


Table 8: Summary of inhibitor effects on 14C bicarbonate uptake into CCMP 1516 and 3268 E. huxleyi cells. Cells were pretreated with inhibitors for 2 hrs prior to pulse labeling.

Inhibitor Target Alkenes PULCA Polar Lipids

1516 3268 1516 3268 1516 3268 Cerulenin FAS-I, II +++ +++ +++ ++ + I

Flufenacet Elongase ++ +++ +++ ++ ++ ++

Platensimycin FAS-II +++ n/a + n/a - n/a

Quizaloflop ACCase +++ +++ +++ +++ +++ +++

“+” denotes degree of inhibition: +++ > 75%, ++ 50-75%, and + 25-50% “I” an increase in carbon flow was seen compared to control. “n/a” no results are available. “-“ inhibitor had no affect, and was comparable to control.



In this study, I set out to determine the flow of carbon into the unusual neutral lipids (PULCAs) and C31-33 cis-alkenes of the haptophyte alga E. huxleyi. I used a combination of techniques, including GC-MS analysis, radiolabeling, and inhibitors, to examine lipid pools during growth cycles, bicarbonate dosing, and light-dark manipulations.

Identification of Neutral Lipids with GC-MS

Through GC-MS analysis I identified PULCA-type C37-38 di and tri- unsaturated methyl ketones, ethyl ketones, methyl esters, and alkenes in lipid extracts of

E. huxleyi cells. All strains also contained C31-34 alkenes. Although I did not confirm stereogeometry of these alkenes, all C37-38 alkenes were previously shown to be trans- unsaturated, while the C31-34 hydrocarbons are cis-unsaturated, and thought to derive from oleic acid, as they have a ω-9 diene bond (Rieley et al 1998). I found that the presence of cis versus trans alkenes was strain dependent (table 4), similar to the results seen by

Conte et al. (1995), who found variations in hydrocarbon types amongst strains. CCMP

3266 the diploid (lithifying) and CCMP 3268 the haploid (flagellated) form of the same cell line, both produced the same lipid classes, but varied in the rate of production.

Another study (Bell & Pond 1996) found that both lipid and fatty acid composition



of coccolith and cell forms have a close similarity, inferring that lipid composition is determined by genotype rather than life cycle stage.

Changes in Neutral Lipid Pools During Growth Conditions

GC-MS analysis showed PULCA production in CCMP 3268 increased dramatically (fig. 8b) following bicarbonate feeding, but the alkenones increased more than the hydrocarbons, so the relative fractions changed significantly. In contrast, for

CCMP 1516, I observed only modest increases in both C31-33 cis alkenes and C37-38

PULCAs after cells were dosed with bicarbonate, and the relative fractions of the dominant C31-33 hydrocarbons and C37-38 methyl ketones remained constant (fig. 8a). This was also supported by pulse-chase labeling of 1516 with 14C bicarbonate, where flow of label into hydrocarbons was similar for both exponential and stationary lipids (fig. 11e, f).

Light-dark experiments showed both hydrocarbon pools responded similarly after bicarbonate dosing. Rontani et al. (2004), observed that cis–alkenes produced by

Chrysotila lamellose (Haptophyta) HAP 17 actually decreased as cells went from early stationary (15 days) to late stationary phase (60 days). Also C31-33 alkenes show a difference in photoreactivity implies a distinct biological syntheses and/or function for the two groups of hydrocarbons (Mouzdahir et al 2001, Rontani et al 1997). It has been suggested based on this information and C31-33 alkenes structure that they are intracellular membrane-bound lipids, and function as regulators of membrane fluidity and rigidity

(Brassell 1993, Prahl et al 1988). However my results show C31-33 alkenes diminish in dark conditions like C37-38 alkenes (fig. 14), mimicking patterns typical of storage lipids.


Acetate Utilization

Since none of my stock cultures were axenic, it is possible that some 14C acetate radiolabeling was due to bacterial uptake. Foreign organisms can readily incorporate the label thus skewing results, and associated with E. huxleyi have been shown to degrade alkenones (Zabeti et al 2010). E. huxleyi cultures are often pre- treated with multiple antibiotics to avoid bacterial contamination (Stern & Tietz 1993).

By treating E. huxleyi cells with kanamycin and ampicillin I hoped to eliminate any bacterial contamination that may affect my results, however I saw no change in 14C acetate uptake (fig. 16b). I also tried differential centrifugation as a means to remove bacteria. E. huxleyi cells are large in comparison to bacterial cells, and will pellet more quickly at low centrifugation speeds. But with centrifugation treatments I also found no change in 14C acetate uptake compared to control (fig. 16b). This leads me to believe 14C acetate uptake comparisons are a reflection of E. huxleyi cells, not contaminates.

I observed that 14C acetate incorporation into E. huxleyi cells occurred in both the light and in the dark (fig. 16a), although dark uptake may have been reduced slightly.

In longer-term pulse-chase experiments, I also observed acetate label move into the ethyl and methyl ketones during the dark (fig. 10f). Similar to these results, Guiheneuf et al.

(2011) observed that 14C acetate labeling of PUFAs in the haptophyte Pavlova lutheri was less sensitive to light intensity than bicarbonate. In contrast, the chlorophyte

Nannochloropsis sp. stopped production of TAGs from acetate in the dark (Schneider &

Roessler 1994). One possible explanation of this is that chlorophytes, which arose from a single endosymbiotic event, are fundamentally photosynthetic organisms and do not have


cytosolic FAS-I systems, thus requiring plastidial FAS for all lipid synthesis. This is supported by recent findings that in the model chlorophyte alga Chlamydomonas, TAGs are actually synthesized entirely in plastids, rather than from FAs exported to endomembrane acyltransferases (Fan et al 2011). In contrast, haptophytes evolved from multiple rounds of endosymbiosis, and are thus hybrid organism combining fundamental

-like’ and ‘plant-like’ metabolic systems, similar to the better-studied diatoms

(Armbrust et al 2004, Bowler et al 2008). My observations support the hypothesis that acetate flow into lipids may occur via cytosolic FAS or elongation systems, independent of light, while bicarbonate fundamentally flows through plastids, and is thus light- dependent. This is also supported by the observations that stationary-phase cells took up acetate at higher rates than exponential cells, while patterns for bicarbonate were opposite

(fig. 10).

Acetate in plant and bacteria models is incorporated either through the plastid in de novo FA synthesis and by elongation elsewhere in the cell (Schneider & Roessler

1994, Thomas 1993). Haptophytes seem to have two distinct enzyme pools involved in

LC-PUFA synthesis, one is intra-chloroplastidic and dependent on light intensity, and the other is extra-chloroplastidic and independent of light (Guiheneuf et al 2011).

Bicarbonate Utilization

In contrast to acetate, bicarbonate acquisition by E. huxleyi was clearly light- dependent. Dark-incubated cells showed no 14C bicarbonate incorporation (fig. 15a), and when cells were transferred to the dark, uptake stopped immediately, only to resume when cells were moved back to the light (fig. 15c). Early studies of bicarbonate


utilization by E. huxleyi, though focused around calcification pathways, found similar results (Paasche 1963, Paasche 1964, Paasche 1966). More recently, Sekino and

Shiraiwa (1994) monitored carbon flow into coccolith precursors and products of photosynthesis, and observed bicarbonate incorporation was light-dependent, except for very brief initial accumulation into internal inorganic carbon pool, which could be explained by residual enzyme function. Tsuji et al (2009) also found 14C bicarbonate fixation in E. huxleyi cells was very low in the absence of light.

I also observed that 14C bicarbonate flowed into both neutral and polar lipids at similar rates (table 5), in contrast to acetate, which went predominantly into polar lipids. Studies have shown that fixed bicarbonate through photosynthesis flows 45-60% into lipid pools (Fernández et al 1994a, Fernández et al 1994b), my pulse chase results were comparable.

Bicarbonate was found to only be incorporated after carbon fixation in the chloroplast through photosynthesis (Schneider & Roessler 1994, Sekino & Shiraiwa


Inhibitor Effects on Neutral Lipid Synthesis

Inhibitor studies of 14C-acetate labeling of CCMP 1516 showed uptake of label into hydrocarbons (C31-33 cis-alkenes) was inhibited by cerulenin and flufenacet; whereas uptake into C37-39-trans-PULCA was inhibited by quizalofop, cerulenin and flufenacet. Inhibitor studies of 14C-bicarboante labeling of CCMP 1516 and 3268 showed no variations between inhibitor behavior; cis-alkenes, trans-alkenes, and trans-

PULCA were inhibited by cerulenin, flufenacet, and quizalofop. Lack of effect of


inhibitors in earlier studies may be attributed to duration of inhibitor application (table 6).

Originally inhibitors were applied for only 30 minutes before pulse labeling, later work showed addition of inhibitors 2 hrs prior to pulse labeling was more effective.

Cerulenin irreversibly inhibits KAS of both multifunctional (homomeric) and unassociated (multimeric) FAS systems (Jaworski et al 1989, MacKintosh et al 1989,

Wakil & Stoop 1983), although cerulenin differentially affects the different KAS in plastidial FAS-II systems, affecting primarily β-ketoacyl-ACP synthase I (KAS I), while acetoacetyl-ACP and β-ketoacyl-ACP synthase II (KAS II) are less sensitive

(MacKintosh et al 1989, Shimakata & Stumpf 1982, Vance et al 1972). I observed strong inhibition by 25 µM cerulenin of lipid synthesis in E. huxleyi, but inhibition was consistently different for labeling from bicarbonate vs. acetate. Cerulenin specifically inhibited incorporation of 14C-bicarbonate into neutral, but not polar lipids, while the opposite was true for 14C-acetate (table 7, 8). The only other known study of cerulenin inhibition on E. huxleyi, Sorrosa (2004), found cerulenin inhibited synthesis of alkenones but had little to no affect on alkenes and polar lipids. The lack of affect on polar lipids might be attriubuted to their structure which not only consits of FA but also sterols, chlorophylls and other membrane lipids which are synthesized by a different pathway.

Therefore, even if the elongation of FA was inhibited by cerulenin as reported (Child &

Shoolingin-Jordan 1998, Funabashi et al 1989, Siggaard-Andersen et al 1991), the percent inhibition should be considerably lower than that of alkenones.

Flufenacet has not been tested on E. huxleyi, but has been found to inhibit higher plant elongases but not FAS complexes in either Arabidopsis ( in the chloroplast)


or yeast (Trenkamp et al 2004). In gracilis flufenacet inhibited mitochondrial fatty acid synthesis but had no affect onfermentation based wax ester synthesis (Tucci et al 2010). Quizaloflop is an herbicide typically applied to crops to inhibit ACCase activity which catalyzes the first step in fatty acid synthesis, but also affects mammals and parasites (Zuther et al 1999). A study on broadleaf grass involving 14C acetate pulse labeling found carbon flow into lipids was inhibited by quizalofop (Bjelk & Monaco


Also separate tests on CCMP 1516 found that 14C bicarbonate labeling of LC cis-alkenes was inhibited by platensimycin, other lipid pools were unaffected as well as

14C acetate incoproation. Plantensimycin is a new synthesized antibiotic, which affects

FAS-II systems in bacteria. However, few studies have been done on the alternative effects of platensimycin (Wang et al 2006).

Summary of Findings

1. CCMP 1323, 1516, and 371 produce C31-33 cis-alkenes, but not C37-39 trans-

alkenes; while CCMP 1742, 3266, and 3268 produce both.

2. External acetate is not acquired by E. huxleyi cells under light dependent

mechanisms and is utilized primarily in production of polar lipids.

3. External bicarbonate acquisition and use in lipid synthesis in E. huxleyi cells is

light-dependent, and as a cellular building block it is distributed more evenly

amongst lipid pools.

4. Flow of carbon into E. huxleyi CCMP 1516 and 3268 cells from external

bicarbonate into lipid pools is inhibited by cerulenin (FAS inhibitor), flufenacet


(elongase inhibitor), and quizaloflop (ACCase inhibitor). Platensiycin (FAS-II

inhibitor) only affects flow of bicarbonate into CCMP 1516 C31-33 cis-alkenes

5. Flow of carbon into E. huxleyi CCMP 1516 cells from external acetate into lipid

pools is inhibited by cerulenin (FAS-I inhibitor). Flufenacet (elongase inhibitor)

and quizaloflop (ACCase inhibitor) only affects flow of acetate into PULCA.

Platensimycin (FAS-II inhibitor) has no effect on the flow of acetate into lipid

pools in CCMP 1516.

Possible PULCA and C31-33 cis-alkene Synthesis Pathways

Using the findings from my study I can support proposed models of lipid synthesis of PULCA and C31-33 cis-alkenes. Bicarbonate is incorporated into initial FA precursors via the Calvin-Benson-Bassham (CBB) cycle in the plastid generating 3- phosphoglycerate (3PGA). 3PGA is used to synthesize pyruvate, which is converted to acetyl-CoA and used by FAS-II (Young et al 2011). Also, pyruvate can be exported to the cytosol to generate cytosolic acetyl-CoA (Thomas 1993). This leaves bicarbonate with two possible fates. Acetate converted into acetyl-CoA can enter through the plastid or the cytoplasm; but I found the majority of acetate is used directly in polar lipids.

From inhibitor studies of cerlunin I confirmed that lipid synthesis is dependent on FAS-I and FAS-II (Thomas 1993), also my findings showed depedence on ACCase for conversion of acetyl-CoA to malonyl-CoA and flow of acetate and bicarbonate into nonpolar lipids. Flufenacet inhibits elongases and drastically affected bicarbonate incorporation in all lipid types, however it had almost no affect on flow of acetate into lipids except for a slight decrese in PULCA; this suggests that elongases are crucial for


the synthesis of early lipid precurosrs and may be cruical later in the addition of acetate to

PULCA. Platensimycin results suggest that LC cis-alkenes are dependent on FAS-II, but

PULCA do not because they showed a very low response to the inhibitor. Since these studies were primarly performed on CCMP 1516, which only generates LC cis-alkenes, we can not confirm the difference in cis-alkene/trans-alkene pathways. Further tests of platensimycin, and 14C acetate of labeling with inhibitors of CCMP 3268 would help clairfy this information. Also by studing incorporation of longer chain 14C labels like palmitate or oleic acid may help in understanding synthesis of these lipids.

Though my resutls were very informative on components involved in the synthesis of PULCA and LC cis-alkenes a definite synthesis pathway can not yet be generated, and I can neither reject or support the models proposed by Rontani et al (2006)

(fig. 3). Since PULCA are highly dependent on bicarbonate, they most likely are synthesized from plastidial components. FAS-II was found unneceessary for their formation, therefore it is possible PULCA originate from C16 precursors from the plastid.

Rontani et al (2006) suggested through the addition of malonyl-CoA to elongate C16, most likely by an elogase, LC precursers to PULCA alkenoates and alkenones are synthesized; my quizaloflop inhibitor findings support this since PULCA syntehsis was impaired by lack of acetyl-CoA to malonyl-CoA conversion. Rontani et al (2006) also suggested that C37-39 trans-alkenes were synthesized from the reduction of C37-39 methylketones, my results however do not dispute or support this.

C31-33 cis-alkenes have a ∆15,22 bonds, suggesting a different pathway

(Rontani et al 2004, Rontani et al 2001, Rontani et al 2006), and the differences I found


between PULCA and LC cis-alkenes in GC-MS analysis and radiolabeling studies however do not support this idea. Rieley et al (1998) hypothesized that LC cis-alkenes were derived from oleic acid, however I can not refute or support this idea without further radio labeling studies. In plant cells LC cis-alkenes, used as wax esters, are generated in the ER and later will be used as structural components (fig. 3b) (Bowsher et al 2008), however C31-33 synthesis shows patterns typical of storage lipids making this model unlikely. Botryococcus braunii, another marine microalgae, produces LC C23-33 cis- alkenes with similar bond arrangments as E. huxleyi’s C31-33 alkenes. Templier et al

(1984) showed oleic acid was directly used in the synthesis of Botryococcus braunii LC cis-alkenes via 14C oleic acid radiolabeling, and then suggested further elongation occurred by the addition of C2 from a source like malonyl-CoA. The mechanism was confirmed by the inhibition of both hydrocarbon and very long chain fatty acid biosynthesis with trichloroacetic acid, which inhibits LC fatty acid synthesis (Templier et al 1987). Inhibition of hydrocarbon synthesis with dithioerythritol was also observed, an inhibitor of decarboxylation, which suggested involvement of a decarboxylation step in the formation of the terminal unsaturation. The finally step, which would require high energy, suggested β-substitution would be implicated, incorporation of 14C labeled β- hydroxy C28 fatty acid into C27 diene was consistent with such a mechanism (Chan Yong et al 1986). I believe this is a more likely mechanism for the synthesis for C31-33 cis- alkenes in E. huxleyi because it utilizes key components also suggsted by Rontani et al

(2006) for the synthesis of PULCA.



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