Thesis

Elucidation of the biosynthetic production pathways of neutral lipids in the marine haptophyte Emiliania huxleyi

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 Isochrysis 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 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 Isochrysis 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.

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