1. An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose William C. Deloache, Zachary N. Russ, Lauren Narcross, Andrew M Gonzales, Vincent J. J. Martin, & John E. Dueber Presented by: Emma Price and Leah Johnston

2. Benzylisoquinoline alkaloids (BIAs) Family of L-tyrosine derived metabolites with a variety of therapeutic uses

3. WHY IS THIS STUDY IMPORTANT? Plant Metabolic Engineering Structures Tools Resources http://labmed.ascpjournals.org/content/41/8/457/F2.expansion.html https://bioweb.uwlax.edu/bio203/s2012/engebos_meag/nutrition.htm

4. Instead of plants? http://yourweeklymicrobe.blogspot.ca/2011/04/yeast-leader-in-libations-king-of.html Shirley Owens, Center for Electron Optics, MSU, 1996 MICROBES! The use of S. cerevisiae in the production of artemisinic acid has already been shown to revolutionize pharmaceutical manufacturing

5. FIRST PART OF THE BIA PATHWAY This part of the BIA pathway occurs efficiently in engineered E. coli Naturally occurring pathway http://www.nature.com/ncomms/journal/v2/n5/full/ncomms1327.html

6. BIA Pathway L -Tyrosine L -DOPA Dopamine4-Hydroxyphenyl-acetaldehyde (s)-Norcoclaurine (s)-Reticuline Codeine Morphine Etc. +

7. S-Reticuline to Morphine (S)-Reticuline Salutaridinol-7-O-acetate CodeinoneCodeine Morphine (R)-Reticuline Salutaridinol SalAT COR1 Many of these steps have been successfully demonstrated in S. cerevisiae

8. E. coli https://en.wikipedia.org/wiki/Carl_Woese http://pubs.acs.org/cen/news/8229/print/8229notw6.html CYTOCHROME P450s (CYPs) 21,000 distinct CYPs Role in the generation of new backbones

9. WHY DO THEY NEED THE FIRST HALF OF THE PATHWAY IN YEAST? Small-scale Large-scale ✔ = E. coli = S. cerevisiae

10. TYROSINE HYDROXYLASES Tyrosine 3-monooxygenases Copper-containing tyrosinases Glucose    L-Tyrosine L-DOPA L-Dopaquinone Melanin 1 2 YEAST DON’T HAVE TYROSINE HYDROXLASES

11. WHAT IS THE ISSUE? Glucose    L-Tyrosine L-DOPA Dopamine BIAs (e.g., morphine) L-DopaquinoneMelanin Betaxanthin DOD DODC #1: Tyrosine hydroxylase 1 2 ✔

12. Glucose    L-Tyrosine L-DOPA Dopamine BIAs (e.g., morphine) L-DopaquinoneMelanin Betaxanthin DOD DODC Cellular fluorescence was measured to determine whether the biosensor activity related accurately to L-DOPA levels produced SOLVING PROBLEM #1: A BIOSENSOR DOD = DOPA dioxygenase

13. ISSUE # 2: Norcoclaurine Synthase (NCS) Solution: Found a suitable, newly identified, NCS in the opium poppy Papaver somniferum DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). Nature Chemical Biology.

14. BIA Pathway L -Tyrosine L -DOPA Dopamine4-Hydroxyphenyl-acetaldehyde (s)-Norcoclaurine (s)-Reticuline Codeine Morphine Etc. +

15. BIA Pathway L -Tyrosine L -DOPA Dopamine4-Hydroxyphenyl-acetaldehyde (s)-Norcoclaurine (s)-Reticuline Codeine Morphine Etc. + *Problem 1 *Problem 2

16. Problem 1 L -Tyrosine L -DOPA Tyrosine Hydroxylase

17. Problem 1 Finding a Yeast-active Tyrosine Hydroxylase Solution Develop an enzyme-coupled biosensor

18. L -Tyrosine L -DOPABetaxanthin Problem 1 Finding a Yeast-active Tyrosine Hydroxylase Yeast active tyrosine hydroxylase DOPA Dioxygenase

19. Problem 1 Finding a Yeast-active Tyrosine Hydroxylase L -Tyrosine L -DOPABetaxanthin Yeast active tyrosine hydroxylase DOPA Dioxygenase Candidate 1: AbPPO2 Candidate 2: CYP76AD1

20. Problem 1 Finding a Yeast-active Tyrosine Hydroxylase DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). Nature Chemical Biology.

21. Problem 1 Improving the Tyrosine Hydroxylase Activity of CYP76AD1 CYP76AD1 Error prone PCR Visual Selection of 17 Highest Betaxanthin producers Top 6 Strains DNA shuffling library

22. Problem 1 Improving the Tyrosine Hydroxylase Activity of CYP76AD1 Double mutant strain of CYP76AD1 DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). Nature Chemical Biology.

23. Problem 1 Finding a Yeast-active Tyrosine Hydroxylase L -Tyrosine L -DOPA Betaxanthin L -Dopaquinone Dopamine Unwanted DOPA oxidase activity

24. Problem 1 Finding a Yeast-active Tyrosine Hydroxylase Tyrosine Hydroxylase with HIGH DOPA oxidase activity Tyrosine Hydroxylase with LOW DOPA oxidase activity Violet Yellow

25. Problem 1 Finding a Yeast-active Tyrosine Hydroxylase DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). Nature Chemical Biology.

26. BIA Pathway L -Tyrosine L -DOPA Dopamine4-Hydroxyphenyl-acetaldehyde (s)-Norcoclaurine (s)-Reticuline Codeine Morphine Etc. + *Problem 1 *Problem 2

27. Problem 2 Putting it all together Dopamine4-Hydroxyphenyl-acetaldehyde (s)-Norcoclaurine (s)-Reticuline Codeine Morphine Etc. + 6OMT CNMT NMCH 4’OMT NCS

28. Problem 2 Putting it all together DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). Nature Chemical Biology.

29. The Big Picture Glucose L- Tyrosine (s)-Reticuline S. Cerevisiae

30. NEXT STEPS (S)-Reticuline titers Identify dopamine transporter Balancing 4-HPAA and dopamine production Tyrosine  (S)-Reticuline (S)-Reticuline  Morphine, etc Put the 2 different pathways into one yeast strain to produce morphine! + tyrosol + 4-HPA DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). Nature Chemical Biology.

31. REFERENCES DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). An enzyme- coupled biosensor enables (S)-reticuline production in yeast from glucose. Nature Chemical Biology, 11, 465-471.