The Biochemistry of DHA Synthesis via a Polyketide Synthase in Schizochytrium

1. The Biochemistry of DHA Synthesis via a Polyketide Synthase in Schizochytrium by Brad Stoehr University of Colorado, Colorado Springs

2. OmegaTech Research • Optimize Docosahexaenoic acid (DHA) yield from Schizochytrium • Locate genes involved in DHA production • Discover the biochemical pathway that Schizochytrium uses to produce DHA • Eventually transfer the genes vital to the biochemical pathway into plants and optimize the harvest

3. Schizochytrium • a thraustochyrid or golden algae • has a large amount of storage triacylglycerols (TAGs), and under suitable conditions DHA is 35-40% of the total fatty acids • DHA-rich oil extracts are intended for use as a nutritional ingredient in foods • determined to be safe, non-toxic, and non-pathogenic

4. It is one of the most abundant compounds in the brain Plays a key role in membrane structure and function Combats cardiovascular disease, cancer, Alzheimer’s, type II diabetes, stroke, depression, pain, schizophrenia, asthma, aging, skin damage, and cystic fibrosis Part of the (n-3) family Functions in membrane receptors, Ca+2& glucose transport, enzymes, metabolism, retinal development, sperm, and membrane fluidity Most recent research is in supplementation Why is DHA so significant?

5. Purpose of this Work! • Human intake of DHA is low • DHA supplementation is one solution • Therefore, we want to expand and optimize supplementation via OmegaTech products - currently have chicken and swine feeds • Optimize products by helping elucidate the biochemical pathway that Schizochytrium uses to synthesize DHA

6. What do we know about Schizochytrium and it’s synthesis of DHA • Contains an FAS I • Contains a Polyketide Synthase (PKS) - found genetically • Pulse-Chase experiments show that radioactivity in DHA fraction w/o an in the other fatty acid fractions

7. Polyketide Synthases (PKSs) • multienzyme systems that make complex natural products • mainly macrolides used as antimicrobials and antifungals • very similar to FASs • structurally, genetically, and enzymatically • use 2-, 3-, & 4-carbon building blocks • decarboxylative condensation is the key reaction • like FASs, there are different types • Type I: large multidomain protein w/ all active sites • Type II:small, discrete enzymes w/ distributed a-sites • Type III: modular PKSs; have multiple copies of a-sites

8. Preliminary Work • Cell Culture • M2B media -vs- M50 media • 1% innoculum @ 30oC • attempted growth curves • Flow Cytometry • used to see if cells are dead or alive • found high viability after ~48hr of incubation

9. Inhibitor Experiments (In vivo) • Inhibitor = Triclosan • an enoyl reductase inhibitor • non-competitive • used as an antibacterial • inserts into type II FASs, but not type I

10. Hypothesis • Schizochytrium’s PKS plays a key role in poly-unsaturated fatty acid (PUFA) synthesis • Triclosan may or may not affect the synthesis of PUFAs, based on its ability to insert into the PKS enzymes. • Triclosan will not affect saturated fatty acid synthesis

11. Significance of Hypothesis • Help determine step-wise synthesis that Schizochytrium utilizes to make PUFAs • Does Schizochytrium utilize FAS by-products (16:0) to make PUFAs • Does Schizochytrium synthesize PUFAs directly via a new pathway, maybe PKS -or-

12. Material and Methods (radioactive) • Cell Culture (M2B media) • Assay • Triclosan incubation • C14-acetate uptake (start) • Incubate for given t • Test tube dip in liquid N2 (stop) • Transmethylation • Thin layer Chromatography (TLC) • Detection

13. Autoradiograph of in vivo C14-labeling of Schizochytrium 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 -t = 5min for lanes 1-6; t = 15min for lanes 11-16 -lane 9 is the superimposed DPA std. -lanes 6, 10, and 16 are negative controls (EtOH)

14. Inhibitor -vs- Liquid Scintillation Count -migration spots were scraped from the plates for t = 15 min -quantitative confirmation of the autoradiograph

15. C14-acetate remaining in the assay supernatant -original amount of radioactivity added was ~ 11x106 cpm

16. Summary of Results • All fatty acid synthesis, both saturated and unsaturated, was increasingly inhibited with increasing [inhibitor] • qualitatively (autoradiograph) • quantitatively (liquid scintillation) • Triclosan does not inhibit uptake of C14-acetate • Duplicate, cold experiments were inconclusive, but showed no real fatty acid synthesis inhibition w/ increasing [Triclosan] and

17. Discussion/Explanations of Results • All fatty acids are made by PKS enzyme • The PKS is susceptible to inhibition • The type I FAS genes are silent • FAS is only turned on during unfavorable circumstances • DHA/DPA is retroconverted back to 16:0 and 18:0 • Tertiary structure of FAS is susceptible to triclosan inhibition • Can only conclude: • triclosan does inhibit all fatty acid production • triclosan does not inhibit C14-acetate uptake

18. More Inhibitor Experiments (In vitro) • Need to determine: • protein extraction process • proper [protein] • proper buffer • proper pH • reaction cocktail • Once above variables are found perform triclosan experiments

19. Protein extract acetone(-20oC) glass bead [PKS] should be active between 0.01-100 ng/mL Tris will make the best buffer Optimum pH will be near 7.0 Cocktail needs will be similar to other organism’s FAS needs sub C14-acetate w/ C14-Mal-CoA Hypotheses

20. Material and Methods (radioactive) • Cell Culture (M2B media) • Extract (acetone powder) • Assay • Change one variable while keeping others constant (e.g. Change [protein] in the same buffer @ the same pH) • Transmethylation • Thin layer Chromatography (TLC) • Detection

21. Autoradiograph from in vitro experiment to determine buffer and protein concentration 1 2 3 4 5 6 7 8 9 10 11 12 13 Tris buffer Mes buffer -lane 6 corresponds to 10l of protein extract and is most active -Hepes buffer experiments not shown

22. Autoradiograph from in vitro experiment to determine optimum pH pH = 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 -Most activity at pH = 7.0 -some activity found at pH = 7.2 and 6.4 -All lanes contain 10l of protein in Tris buffer

23. Summary • 10l is most active [protein] - corresponds to ~6ng/mL of PKS, assuming 1% of total protein • Tris is the most active buffer • pH = 7.0 is most active Now we can keep these constant and vary the assay cocktail

24. Constant Assay Cocktails • Cocktail A: • 50mM NADH, 60mM NADPH, 500mM DTT, 5mM acetyl CoA, and 10mM Mal-CoA (half hot/half cold) • used for [protein], buffer, and pH determination experiments • Cocktail B: • 500mM DTT, 5mM acetyl-CoA,10l of protein extract in Tris (pH=7.0), and 10mM Mal-CoA (half hot/half cold)

25. Hypothesis • The assay will require a reduced nicotinamide compound, but which it will favor is unknown • ATP should have a stimulatory effect, because high energy leads to storage pathways • If PKS or FAS is using a desaturase it will require oxygen • Triclosan should show similar inhibitory effect as in vivo experiments

26. Autoradiograph showing initial NADH/NADPH in vitro experiment 1 2 3 4 5 6 7 8 9 10 11 12 NADH NADPH -lane 10 = 1.0 mM NAD+ -lane 11 = 1.0 mM NADP+ -lane 12 has no nicatinamide

27. Autoradiograph showing confirmation of NADH/NADPH experiment, w/ initial NAD+in vitro experiment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 NADH NADPH NAD+ -lane 10 contains no nicatinamide -lane 11 = 1.0 mM NAD+ -lane 12 = 1.0 mM NADP+

28. Duplicate experiment, except 2mM of ATP and 2mM of Mg+2 was added to each assay 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 NADH NADPH NAD+ -lane 10 contains no nicatinamide -lane 11 = 1.0 mM NAD+ -lane 12 = 1.0 mM NADP+

29. Autoradiograph showing NAD+ confirmation and initial Triclosan experiment (in vitro) 1 2 3 4 5 6 7 8 9 10 11 12 NAD+ Triclosan (w/ 1.0mM NAD+) -lane 3 = 1.0 mM NAD+ -lane 9 = 1.0 mg/mL of triclosan -lane 11 = 0.1 mg/mL of triclosan

30. Summary of Results • NADH in assay cocktail shows no activity • NADPH shows slight activity @ 0.5 and 2.5mM • NADP+ shows slight activity @ 1.0mM • NAD+ shows the most activity of all @ 1.0mM • ATP/Mg+2 shows causes less activity • Anaerobic experiments showed no activity (data not shown) • In vitro triclosan experiments do not match in vivo experiments • Cold experiments offer no support

31. Discussion • De novo fatty acid synthesis is occurring • Mal-CoA cannot cross membranes • Is it PKS or FAS? Both? • Nicatinamide results are surprising • NAD+ is more active than both NADH and NADPH • May be due to physiological pH or DTT addition • Could be that Schizochytrium’s PKS is truly novel • ATP may actually inhibit PKS • O2 is required for fatty acid synthesis • suggests that a desaturase is involved • may help facilitate a NAD+-rich environment • In vitro triclosan experiments need further investigation

32. Conclusions • Schizochytrium is extremely viable after 48 hours of incubation using a 1% inoculum • Triclosan inhibits all fatty acid synthesis in vivo • Triclosan does not inhibit Schizochytrium’s ability to uptake acetate readily • The pH must be at or near 7.0 for in vitro experimentation • The presence of ATP hinders the enzymes ability to synthesize fatty acids • Molecular oxygen is required for DHA production

33. Acknowledgements • OmegaTech • Dr. D. Guerra • Dr. R. Melamed • Dr. M. Newell • Dr. R. Pyati • Dr. S. Berry-Lowe • Ruth Jackson